Note : Les descriptions sont présentées dans la langue officielle dans laquelle elles ont été soumises.
WO 2021/163042
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PREPARING AND MODIFYING MEROTERPENE POLYKETIDES, KETONES, AND
LACTONES FOR CANNABINOID SEMISYNTHESIS
STATEMENT ABOUT FEDERAL FUNDING
Not applicable.
CROSS REFERENCE TO RELATED APPLICATION
This application claims priority to US Provisional application nos. US
62/975,378
filed February 12, 2020; US 63/019,098 filed May 1, 2020; and US 63/122,360
filed
December 7, 2020 each of which is incorporated herein in its entirety by
reference.
REFERENCE TO A SEQUENCE LISTING
The instant application contains a Sequence Listing, which has been submitted
electronically in ASCII format and is hereby incorporated by reference in its
entirety. Said ASCII copy, created on February 8, 2021, is named LYGOS-0040-01-
WO SL.txt and is 11,773 bytes in size.
FIELD
This invention relates at least in part to processes, including semi-
synthetic, and
synthetic processes for preparing meroterpene polyketides, ketones, and
lactones, such
as cannabinoids, and meroterpene compositions provided thereby.
BACKGROUND
There is a need for processes, particularly semi-synthetic, and synthetic
processes
for preparing cannabinoids, and cannabinoid compositions provided thereby.
SUMMARY
In certain aspects, provided herein are processes, including semi-synthetic,
and
synthetic processes for preparing cannabinoids, and cannabinoid compositions
provided
thereby. A semi-synthetic process refers to a process of preparing one or more
cannabinoids, where a fermentation-based process is combined, preferably but
not
necessarily without separation, purification, solvent-swap, and the likes,
with a chemical
synthesis process.
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In one aspect, provided herein is a process for preparing one or more of a
compound of formula (IA), (113), and (IC):
OHHO 0 0
40 p 0
HO R3 HO R4
IA IB IC
or a salt or an ester (carboxy and/or phenolic) thereof, wherein
Ri is H or CO2H;
each R2, R3, and R4 is independently C3-Cio alkyl, 03-010 alkenyl, or 03-Ci0
alkynyl,
preferably, 03-08 alkyl, more preferably, n-pentyl or n-propyl;
the process comprising:
fermenting a recombinant microorganism comprising: a polyketide synthase,
wherein the polyketide synthase combines an acyl-CoA and two or more, such as
two or
three, malonyl-CoA to produce a polyketide thereby preparing one or more of a
compound
of formula (IA), (IB), and (IC) or the salt or the ester thereof. Optionally a
dimeric a-F13
barrel (DABB) protein is also co-expressed with the polyketide resulting in a
polyketide
comprising a carboxylic acid. In one embodiment, a compound of formula IA
comprises
aromatic polyketides (Ri=H). In one embodiment, a compound of formula IA
comprises
aromatic polyketides (R1=CO2H).
In one embodiment, the microorganism is fermented aerobically in the presence
of a water immiscible, liquid, hydrophobic phase, which dissolves the one or
more of a
compound of formula (IA), (IB), and (IC) or the salt or ester thereof. In
another
embodiment, the process further comprises separating the hydrophobic phase
from an
aqueous phase comprising the microorganism, the separating comprising a first
continuous centrifugation to separate the cells and a bulk of a spent broth
from the
hydrophobic phase, followed by a second continuous centrifugation to separate
the
hydrophobic phase from the remaining aqueous phase. In another embodiment, the
process further comprises: esterifying, isoprenylating, or performing an
annulation of the
compound included in the hydrophobic phase, under conditions suitable to
perform an
esterification, isoprenylation, or annulation without the need for a solvent
swap.
In another aspect, provided herein is a process comprising:
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aerobically fermenting a recombinant microorganism comprising: a polyketide
synthase, optionally an olivetolic acid cyclase (OAC), and further optionally
a hexanoyl
Co-A synthetase (HCS), wherein the fermenting is performed in the presence of
a water
immiscible, liquid, hydrophobic phase,
to prepare one or more of: olivetolic acid or a salt or ester thereof, and
olivetol or
an ester thereof,
wherein the hydrophobic phase dissolves olivetolic acid or a salt or ester
thereof
or olivetol or an ester thereof.
In another aspect, provided herein is a process comprising:
aerobically fermenting a recombinant microorganism comprising: a polyketide
synthase, optionally an olivetolic acid cyclase (OAC), and further optionally
butyryl Co-A
synthetase, wherein the fermenting is performed in the presence of a water
immiscible,
liquid, hydrophobic phase;
to prepare one or more of: divarinic acid or a salt or ester thereof, and
divarin,
wherein the hydrophobic phase dissolves divarinic acid or a salt or ester
thereof or
divarin, as they are prepared.
In some embodiments, the microorganism comprises an olivetolic acid cyclase
(OAC). In some embodiments, the microorganism comprises a hexanoyl Co-A
synthetase
(HCS). In other embodiments, a variety of acyl activating enzymes, which are
well known
to the skilled artisan, other than HCS or CsAAE1, are useful in accordance
with this
invention.
In one embodiment, 3 acyl -CoAs are combined.
In one embodiment, a compound of formula IA is provided. In one embodiment, a
compound of formula IB is provided. In one embodiment, a compound of formula
IC is
provided.
In one embodiment, each R1 independently is H. In one embodiment, each R1
independently is CO2H or a salt thereof.
In one embodiment, each R2 is independently C3-C10 alkyl. In one embodiment,
each R2 is independently C3-C8 alkyl. In one embodiment, each R2 is
independently octyl.
In one embodiment, each R2 is independently pentyl. In one embodiment, each R2
is
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independently C3-Cio propyl. In one embodiment, each R2 is independently C3-
Cio
alkenyl. In one embodiment, each R2 is independently C3-Cio alkynyl.
In one embodiment, each R3 is independently C3-C10 alkyl. In one embodiment,
each R3 is independently C3-C8 alkyl. In one embodiment, each R3 is
independently octyl.
In one embodiment, each R3 is independently pentyl. In one embodiment, each R3
is
independently C3-C10 propyl. In one embodiment, each R3 is independently C3-
C10
alkenyl. In one embodiment, each R3 is independently C3-C10 alkynyl.
In one embodiment, each R4 is independently C3-Cio alkyl. In one embodiment,
each R4 is independently C3-C8 alkyl. In one embodiment, each R4 is
independently octyl.
In one embodiment, each R4 is independently pentyl. In one embodiment, each R4
is
independently C3-Clo propyl. In one embodiment, each R4 is independently C3-
Clo
alkenyl. In one embodiment, each R4 is independently C3-Clo alkynyl.
In some embodiments, the alkyl, alkenyl, or alkynyl groups are substituted
with 1 -
3 substituents. Suitable substituents include halo, hydroxy, vinyl, ethynyl,
and the likes.
BRIEF DESCRIPTION OF FIGURES
Figure 1 schematically illustrates recovery of olivetol and other compounds of
formula IA in accordance with the present invention.
Figure 2 schematically illustrates semisynthesis of cannabinoids (CBG) by
prenylation of fermented olivetol.
Figure 3 schematically illustrates semisynthesis of cannabinoids (CBC) by
prenylation of fermented olivetol.
DETAILED DESCRIPTION
While the present invention is described herein with reference to aspects and
specific embodiments thereof, those skilled in the art will recognize that
various changes
may be made and equivalents may be substituted without departing from the
invention.
The present invention is not limited to particular nucleic acids, expression
vectors,
enzymes, host microorganisms, or processes, as such may vary. The terminology
used
herein is for purposes of describing particular aspects and embodiments only,
and is not
to be construed as limiting. In addition, many modifications may be made to
adapt a
particular situation, material, composition of matter, process, process step
or steps, in
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accordance with the invention. All such modifications are within the scope of
the claims
appended hereto.
Definitions
As used in the specification and the appended claims, the singular forms "a",
"an",
and "the" include plural referents unless the context clearly dictates
otherwise. Thus, for
example, reference to an "expression vector" includes a single expression
vector as well
as a plurality of expression vectors, either the same (e.g., the same operon)
or different;
reference to "cell" includes a single cell as well as a plurality of cells;
and the like.
As used herein, the term "express", when used in connection with a nucleic
acid
encoding an enzyme or an enzyme itself in a cell, means that the enzyme, which
may be
an endogenous or exogenous (heterologous) enzyme, is produced in the cell. The
term
"overexpress", in these contexts, means that the enzyme is produced at a
higher level,
i.e., enzyme levels are increased, as compared to the wild type, in the case
of an
endogenous enzyme. Those skilled in the art appreciate that overexpression of
an
enzyme can be achieved by increasing the strength or changing the type of the
promoter
used to drive expression of a coding sequence, increasing the strength of the
ribosome
binding site or Kozak sequence, increasing the stability of the mRNA
transcript, altering
the codon usage, increasing the stability of the enzyme, and the like.
The terms "ferment", "fermentative", and "fermentation" are used herein to
describe
culturing host cells and microbes under conditions to produce useful
chemicals, including
but not limited to conditions under which microbial growth, be it aerobic or
anaerobic,
occurs.
The terms "cell," "host cell" "microorganism" and "host microorganism" are
used
interchangeably herein to refer to a living cell that can perform one or more
steps of the
cannabinoid pathway, e.g., as illustrated herein below. A host cell can be (or
is)
transformed via insertion of an expression vector. A host microorganism or
cell as
described herein may be a prokaryotic cell (e.g., a microorganism of the
kingdom
Eubacteria) or a eukaryotic cell. As will be appreciated by one of skill in
the art, a
prokaryotic cell lacks a membrane-bound nucleus, while a eukaryotic cell has a
membrane-bound nucleus.
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The terms "isolated" or "pure" refer to material that is substantially, e.g.
greater
than 50% or greater than 75%, or essentially, e.g., greater than 90%, 95%, 98%
or 99%,
free of components that normally accompany it in its native state, e.g., the
state in which
it is naturally found or the state in which it exists when it is first
produced.
Polyketide synthases (PKSs) are a family of multi-domain enzymes or enzyme
complexes that produce polyketides, a large class of secondary metabolites, in
bacteria,
fungi, plants, and a few animal lineages. The terms "polyketide synthase",
"PKS", "olivetol
synthase" ("OLS"), "tetraketide synthase", TKS, and olivetolic synthase as
described
herein or elsewhere typically refers to any enzyme capable of converting three
molecules
of malonyl-CoA and one molecule of hexanoyl-CoA to olivetol. A wild type
example of an
OLS is the native C. sativa OLS enzyme (UniProt ID: B1Q2B6; SEQ ID NO: 1).
Sequence ID 1: TKS
MNHLRAEGPASVLAIGTANPENILIQDEFPDYYFRVTKSEHMTQLKEKFRKICDKSMIRK
RNCFLNEEFILKONPRLVEHEMOTLDARODNALVVEVPKLGKDACAKAIKEWGQPKSKI
THLIFTSASTTDMPGADYFICAKLLGLSPSVKRVMMYQLGCYGGGTVLRIAKDIAENNK
GARVLAVCCDIMACLFRGPSDSDLELLVGQAIFGDGAAAVIVGAEPDESVGERPIFELV
STGQTILPNSEGTIGGHIREAGLIFDLHKDVPMLISNNIEKCLIEAFTPIGISDWNSIFWITH
PGGKAILDKVEEKLDLKKEKFVDSREIVLSEFIGNMSSSTVLFVMDELRKRSLEEGKSTT
GDGFEWGVLFGFGPGLTVERVVVRSVPIKY
The term "hexanoyl-CoA synthetase" ("HCS") as used herein refers to any enzyme
capable of catalyzing the conversion of hexanoate (a short-chain fatty acid
anion that is
the conjugate base of hexanoic acid, also known as caproic acid) or hexanoic
acid, and
a free CoA to hexanoyl-CoA. A non-limiting example of a hexanoyl-CoA
synthetase is the
FadK protein derived from E. coll.
The cannabinoid biosynthetic pathway utilizes a variety of enzymes, catalysts,
and
intermediate compounds. For example, cannabigerolic acid synthase (EC:
2.5.1.102) is
used to convert OLA to cannabigerolic acid, which is a key intermediate acted
upon by a
variety of enzymes during THC synthesis. Cannabidiolic acid synthase (EC:
1.21.3.7) is
used to convert cannabigerolic acid into cannabidiolic acid.
Tetrahydrocannabinolic acid
synthase (EC: 1.21.3.8) is used to convert cannabigerolic acid into A9-
tetrahydrocannabinolic acid. A cannabichromenic acid synthase is used to
convert
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cannabigerolic acid into cannabichromenic acid. These three olivetolic acid-
derived
compounds (i.e., cannabidiolic acid, A9-tetrahydrocannabinolic acid, and
cannabichromenic acid) are themselves converted to even more diverse
cannabinoids
via a combination of oxidation, decarboxylation, and isomerization reactions,
which can
be catalyzed using either biological or synthetic catalysts, or can also occur
spontaneously following heating and/or application of UV light. For example,
cannabidiol
results from cannabidiolic acid decarboxylation, A9-tetrahydrocannabinol
results from A9-
tetrahydrocannabinolic acid decarboxylation, and subsequent isomerization of
A9-
tetrahydrocannabinol results in A6-tetrahydrocannabinol.
The term "optional" or "optionally" as used herein mean that the subsequently
described feature or structure may or may not be present, or that the
subsequently
described event or circumstance may or may not occur, and that the description
includes
embodiments where a particular feature or structure is present and embodiments
where
the feature or structure is absent, or embodiments where the event or
circumstance
occurs and embodiments where it does not.
A non-limiting illustration of the cannabinoid pathway is included in the
scheme
below.
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Descriptive Embodiments
In one aspect, provided herein is a process for preparing one or more of a
compound of formula (IA), (16), and (IC):
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OH 0 0
I
HO R2
HO R3 HO R4
RI
IA IB IC
or a salt or an ester (carboxy and /or phenolic) thereof, wherein
R1 is H or CO2H;
each R2, R3, and R4 is independently C3-C10 alkyl, C3-Cio alkenyl, or C3-C10
alkynyl,
preferably, C3-C8 alkyl, more preferably, n-pentyl or n-propyl;
the process comprising:
fermenting a recombinant microorganism comprising: a polyketide synthase,
wherein the
polyketide synthase combines an acyl-CoA and two or more, such as two or
three,
malonyl-CoA to produce a polyketide thereby preparing one or more of a
compound of
formula (IA), (IB), and (IC) or the salt or the ester thereof. Optionally a
dimeric a-F13 barrel
(DABB) protein is also co-expressed with the polyketide resulting in a
polyketide
comprising a carboxylic acid.
In one embodiment the ester is independently a carboxylic acid ester, or in
other
words, the carboxylic acid moiety corresponding to R1 is esterified.
In another
embodiment, the ester is independently a phenolic ester.
In another embodiment, at least one compound prepared is of formula (IA).
Without being bound by theory, the polyketide synthase combines an acyl-CoA
and three
malonyl-CoA to prepare a compound of formula IA, where Ri¨ H.
In another embodiment, at least one compound prepared is of formula (IB).
In another embodiment, at least one compound prepared is of formula (IC).
In another embodiment, one or more phenolic hydroxy moieties of the compound
of formula (IA), (113), or (IC), or a salt thereof is esterified in vivo (or
endogenously) as a
result of overexpression of an arylesterase in the microorganism.
In another embodiment, the compound of formula (IA), (IB), or (IC) is
glycosylated
in vivo as a result of overexpression of a glycosylase in the microorganism.
In another embodiment, the microorganism is a fungus. In another embodiment,
the microorganism is a bacteria. In another embodiment, the microorganism is
an algae.
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In another embodiment, the microorganism is yeast. In another embodiment, the
microorganism is S. cerevisiae.
In some embodiments, the microorganism is a prokaryotic organism. In some
embodiments, the microorganism is an eukaryotic organism. In some embodiments,
the
microorganism is a fungal organism. In some embodiments, the microorganism is
a yeast
organism. In some embodiments, the microorganism is a bacterial organism In
some
embodiments, the microorganism is a unicellular organism. In some embodiments,
the
microorganism is a bacterial cell. In some embodiments, the microorganism is
an
eukaryote. In some embodiments, the microorganism is a yeast cell. In various
embodiments, the yeast is selected from the non-limiting list of example
genera: Candida,
Cryptococcus, Hansenula, Issatchenkia, Kluyveromyces, Komagataella, Lipomyces,
Pichia, Rhodosporidium, Rhodotorula, Saccharomyces or Yarrowia. In some
embodiments, the microorganism is a fungus. In some embodiments, the
microorganism
is an algae. In some embodiments, the microorganism is a P. kudriavzevii
organism. In
some embodiments, the microorganism is a P. pastoris organism. In some
embodiments,
the microorganism is a S. cerevisiae organism. In some embodiments, the
microorganism
is a Y. /ipo/ytica organism. In some embodiments, the microorganism is a
Kluyveromyces
marxianus organism.
In some embodiments, the microorganism is a bacterial cell. In some
embodiments, the microorganism is a bacterial cell selected from Bacillus,
Clostridium,
Corynebacterium, Escherichia, Pseudomonas, and Streptomyces. In some
embodiments, the microorganism is an E. co//organism.
As is apparent to the skilled artisan, the microorganisms disclosed herein are
host
cells for the purpose of this invention.
In another embodiment, the microorganism is fermented aerobically in the
presence of a water immiscible, liquid, hydrophobic phase which dissolves the
one or
more of a compound of formula (IA), (IB), and (IC) or the salt or ester
thereof. In another
embodiment, the process further comprises separating the hydrophobic phase
from an
aqueous phase comprising the microorganism, the separating comprising a first
continuous centrifugation to separate the cells and a bulk of a spent broth
from the
hydrophobic phase, followed by a second continuous centrifugation to separate
the
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hydrophobic phase from the remaining aqueous phase. In another embodiment, the
process further comprises: esterifying, isoprenylating, or performing an
annulation of the
compound included in the hydrophobic phase, under conditions suitable to
perform an
esterification, isoprenylation, or annulation without the need for a solvent
swap. In
another embodiment, the compound prepared is isoprenylated. In another
embodiment,
the compound prepared is esterified. In another embodiment, the compound
prepared is
made to undergo an annulation.
In certain embodiments, carbon feedstocks are utilized for production of
olivetol or
another compound produced herein. Suitable carbon sources include, without
limitation,
those selected from the group consisting of purified sugars (e.g., dextrose,
sucrose,
xylose, arabinose, lactose, etc.); plant-derived, mixed sugars (e.g.,
sugarcane, sweet
sorghum, molasses, cornstarch, potato starch, beet sugar, wheat, etc.), plant
oils, fatty
acids, glycerol, cellulosic biomass, alginate, ethanol, carbon dioxide,
methanol, and
synthetic gas ("syn gas").
In some embodiments, one or multiple intermediates and precursors of the
cannabinoid pathways, including sugar, an acid of formula R2-CO2H or a salt
thereof,
malonic acid, hexanoic acid, mevalonate, olivetol, and olivetolic acid are
employed as a
feedstock. In some embodiments, an acid of formula R2-CO2H or a salt thereof
is
employed as a feedstock. In one embodiment the one or multiple intermediates
and
precursors of the cannabinoid pathway are utilized in a cell free system,
e.g., and without
limitation, with a prenyl transferase that condenses olivetol/olivetolic acid
and GPP. In
some embodiments, another enzyme of the cannabinoid pathway is incorporated in
a
host cell (or added to a cell free system) to further process CBGA or CBG into
THCA,
CBDA, THC, CBD or other CBG(A) derivative. Without being bound by theory, in
some
embodiments, such a feedstock would result in a commercially relevant process
without
the limitations and timelines associated with careful balancing of full
pathway enzymes in
a cell or cell-free system.
A given host cell may catabolize a particular feedstock efficiently or
inefficiently. If
a host cell inefficiently catabolizes a feedstock, then one can modify the
host cell to
enhance or create a catabolic pathway for that feedstock. Additional
embodiments of the
invention include the use of methanol catabolizing host strains. In some
embodiments,
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the host is a yeast strain. In some embodiments, the host is selected from S.
cerevisiae,
Pichia kudriavzevii, Komagataella pastoris, Pichia methanol/ca, or Pichia
pastor/s.
The invention utilizes microorganisms and host cells comprising genetic
modifications
that increase titer, yield, and/or productivity of olivetol or another
compound produced
herein through the increased ability to catabolize non-native carbon sources.
Wild type S.
cerevisiae cells are unable to catabolize pentose sugars, lignocellulosic
biomass, or
alginate feedstocks. In some embodiments, the invention provides a S.
cerevisiae cell
comprising a heterologous nucleic acid encoding enzymes enabling catabolism of
pentose sugars useful in production of olivetol, as described herein. In other
embodiments, the heterologous nucleic acid encodes enzymes enabling catabolism
of
lignocellulosic feedstocks. In yet other embodiments of the invention, the
heterologous
nucleic acid encodes enzymes increasing catabolism of alginate feedstocks.
In another embodiment, the compound dissolved in the hydrophobic phase is one
or both of olivetolic acid or a salt thereof and olivetol.
In another aspect, provided herein is a process comprising:
aerobically fermenting a recombinant microorganism comprising: a polyketide
synthase, optionally an olivetolic acid cyclase (OAC), and further optionally
a hexanoyl
Co-A synthetase (HCS), wherein the fermenting is performed in the presence of
a water
immiscible, liquid, hydrophobic phase,
to prepare one or more of: olivetolic acid or a salt or ester thereof, and
olivetol or
an ester thereof,
wherein the hydrophobic phase dissolves olivetolic acid or a salt or ester
thereof
or olivetol or an ester thereof.
In another embodiment, the olivetolic acid is partially or completely
esterified
endogenously within the microorganism to prepare the olivetolic acid ester.
In another embodiment, the olivetolic acid ester is prepared exogenously
comprising esterifying olivetolic acid with an alcohol under conditions
suitable to prepare
an olivetolic acid ester.
The esterification can be performed in presence of suitable esterification
catalyst,
as is well known to the skilled artisan. In some embodiments the
esterification catalyst is
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soluble in the hydrophobic phase utilized herein, and partitions partially or
completely into
the hydrophobic phase.
In another embodiment, one or more hydroxyl moieties of olivetolic acid,
olivetol,
or an olivetolic acid ester is partially or completely glycosylated by the
microorganism to
provide glycosylated olivetolic acid, glycosylated olivetol, or glycosylated
olivetolic acid
ester. In some embodiments, the glycosylating microorganism overexpresses
glycosylation enzymes. In some embodiments, the glycosylating
microorganism
overexpresses enzymes producing UDP-glucose.
In some embodiments, hexanoic acid or a salt of each thereof is exogenously
supplied to a reactor where the fermenting occurs. In some embodiments, 3-
oxooctanoic
acid, or a salt of each thereof is exogenously supplied to a reactor where the
fermenting
occurs. In some embodiments, 3,5-dioxodecanoic acid or a salt of each thereof
is
exogenously supplied to a reactor where the fermenting occurs. In some
embodiments,
3,5,7-trioxododecanoic acid or a salt of each thereof is exogenously supplied
to a reactor
where the fermenting occurs.
In one embodiment, the process further comprises separating the hydrophobic
phase from an aqueous phase, the separating comprising a first continuous
centrifugation
to separate the cells and a bulk of a spent broth from the hydrophobic phase,
followed by
a second continuous centrifugation to separate the hydrophobic phase from the
remaining
aqueous phase. In one embodiment, the process further comprises isoprenylating
the
olivetol, olivetolic acid or a salt thereof, or the olivetolic acid ester
included in the
hydrophobic phase, without the need for a solvent swap, under conditions
suitable to
perform an isoprenylation, to prepare a cannabinoid or a mixture of
cannabinoids.
In another embodiment, the olivetolic acid or the salt thereof contained in
the
hydrophobic phase is esterified with an alcohol under conditions suitable to
esterify the
carboxyl moiety of olivetolic acid or a salt thereof to yield alkyl
olivetolate. In another
embodiment, the alcohol is selected from alcohols with 2 or more carbons such
as C2-C8
alcohols.
In another embodiment, the alkyl olivetolate is reacted with an isoprenoid, or
is
isoprenylated, to produce a cannabinoid. In some embodiments, the reaction is
catalyzed
by a Bronsted acid. In some embodiments, the reaction is catalyzed by a Lewis
acid.
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Examples of suitable catalysts include without limitation organic acids (e.g.
trifluoroacetic
acid, methanesulfonic acid, tosic acid, and the likes), mineral acids or
solutions of mineral
acids (e.g. hydrochloric acid, nitric acid, sulfuric acid, and the likes),
polymer-supported
acids (e.g. Amberlyst-15, polymer-supported tosic acid, Lewis acids (e.g. BF3,
Sc(0Tf)3,
and the likes), amino acids, or organocatalysts.
In another embodiment, the cannabinoid or the cannabinoid mixture comprises a
carboxyl moiety or a salt thereof, and is decarboxylated under conditions
suitable for
decarboxylation, to prepare a decarboxylated cannabinoid. The decarboxylation
can be
modulated by heating the solution and/or by addition of a catalyst and/or by
the addition
of a base.
In another embodiment, the olivetolic acid or the salt thereof contained in
the
hydrophobic phase is decarboxylated under conditions suitable for
decarboxylation to
provide an initial composition comprising olivetol. An acid may be added
before or during
decarboxylation to modulate the decarboxylation reaction. The reaction mixture
may be
heated to increase the decarboxylation rate. A base may be added to modulate
the
decarboxylation reaction.
In another embodiment, the initial composition comprising olivetol is
isoprenylated
under conditions suitable for isoprenylating a phenolic compound. Examples of
compounds useful in isoprenylating include without limitation geraniol,
farnesol,
geranylgeraniol, p-mentha-2,8-dien-1-ol (or (1S,4R)-p-Mentha-2,8-dien-1-ol),
citral, and
the likes. In some embodiments, the isoprenylated compound is cannabigerol
(CBG). In
another embodiment, the isoprenylated compound is cannabigerolic acid (CBGA).
In some embodiments, the initial composition comprising olivetol is reacted
with
geraniol and an acid under conditions suitable to undergo a Friedel-Crafts
alkylation to
provide cannabichromene. The reaction may be performed in a suitable solvent,
e.g., a
solvent that is inert to the reactants and the reagents. In some embodiments,
a Bronsted
acid is employed. In some embodiments, a Lewis acid is employed. The acid can
be
used in catalytic amounts. In some embodiments, the isoprenylated compound is
cannabigerol (CBG). In some embodiments, the isoprenylated compound
is
cannabigerolic acid (CBGA). Suitable acids include a Bronsted acid such as a
sulfonic
acid, such as p-toluene sulfonic acid, or a Lewis acid such as BF3 etherate,
and the likes.
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Methods for reacting geraniol with olivetol are known in the art, which can be
modified by
the skilled artisan based on the present disclosure to provide cannabigerol as
provided
herein. See, e.g., J. Biol. Chem., Vol. 271, No. 29, Issue of July 19, pp.
17411-17416,
1996 (incorporated herein by reference). Unreacted olivetol may be separated
by one or
more or crystallization and chromatography.
In some embodiments, the initial composition comprising olivetol is reacted
with
citral under conditions suitable to undergo cyclization to provide
cannabichromene (CBC).
The reaction may be performed in a suitable solvent, e.g., a solvent that is
inert to the
reactants and the reagents. In some embodiments, the isoprenylated compound is
cannabichromene (CBC). In some embodiments, the isoprenylated compound is
cannabichromic acid (CBCA). In some embodiments, citral reacts with olivetol
under
basic conditions to provide cannabichromene. Suitable bases include a primary
amine,
such as without limitation propyl amine and tertiary butyl amine; pyridine;
and the likes.
Methods for reacting citral with olivetol are known in the art, which can be
modified by the
skilled artisan based on the present disclosure to provide cannabichromene as
provided
herein. See, e.g., J. Heterocyclic Chem., volume15, Issue 4,1978, pages 699-
700 and
US Pat. No. 4,315,862 (each incorporated herein by reference). Unreacted
olivetol may
be separated by washing with alkali such as NaOH and the likes, or by
chromatography
with alkali impregnated silica.
In another embodiment, the cannabinoid composition is purified, optionally
hydrolyzed, and isolated to provide one or more cannabinoids Hydrolysis may
precede or
follow purification. In another embodiment, the total cannabinoids contained
in the
isolated product is at least 25%, or 50%, or 75%, or 90%, or 95%, or 98%, or
99% of a
single cannabinoid. In some embodiments, after purification, the remaining
amount may
include one or more of a different regioisomer, a different enantiomer, a
different
diastereomer, or a solvent
In another embodiment, the cannabinoid is cannabigerolic acid (CBGA). In
another embodiment, the cannabinoid is cannabichromenic acid (CBCA). In
another
embodiment, the cannabinoid is cannabinolic acid (CBNA). In another
embodiment, the
cannabinoid is tetrahydrocannabinoic acid (THCA). In another embodiment, the
cannabinoid is cannabidiolic acid (CBDA). In another embodiment, the
cannabinoid is
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cannabigerol (CBG). In another embodiment, the cannabinoid is cannabichromene
(CBC). In another embodiment, the cannabinoid is cannabinol (CBN). In another
embodiment, the cannabinoid is tetrahydrocannabinol (THC). In another
embodiment,
the cannabinoid is cannabidiol (CBD). In another embodiment, the cannabinoid
is a
prenylogous version of the above (e.g. sesqui-CBG). In another embodiment, the
cannabinoid is a compound that causes activation of the CB1 , CB2, or TRP
receptor.
In another aspect, provided herein is a process comprising:
aerobically fermenting a recombinant microorganism comprising: a polyketide
synthase, optionally an olivetolic acid cyclase (OAC), and further optionally
butyryl Co-A
synthetase, wherein the fermenting is performed in the presence of a water
immiscible,
liquid, hydrophobic phase;
to prepare one or more of: divarinic acid or a salt or ester thereof, and
divarin,
wherein the hydrophobic phase dissolves divarinic acid or a salt or ester
thereof or
divarin, as they are prepared.
In one embodiment. the divarinic acid is partially or completely esterified
endogenously within the microorganism to prepare the divarinic acid ester. The
OH and/or
the CO2H can be esterfied.
In another embodiment, the divarinic acid ester is prepared exogenously
comprising esterifying olivetolic acid with an alcohol under conditions
suitable to esterify
a carboxylic acid.
The esterification can be performed in presence of suitable esterification
catalyst,
as is well known to the skilled artisan. In some embodiments the
esterification catalyst is
soluble in the hydrophobic phase utilized herein, and partitions partially or
completely into
the hydrophobic phase.
In another embodiment, one or more hydroxyl moieties of divarinic acid,
divarin, or
divarinate esters are partially or completely glycosylated by the
microorganism to provide
glycosylated divarinic acid or a salt thereof, glycosylated divarin, or
glycosylated
divarinate ester. In some embodiments, the glycosylating microorganism
overexpresses
glycosylation enzymes. In some embodiments, the glycosylating
microorganism
overexpresses enzymes producing UDP-glucose.
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In some embodiments, butyric acid or a salt of each thereof is exogenously
supplied to a reactor where the fermenting occurs. In some embodiments, 3-
oxooctanoic
acid, or a salt of each thereof is exogenously supplied to a reactor where the
fermenting
occurs. In some embodiments, 3,5-dioxodecanoic acid or a salt of each thereof
is
exogenously supplied to a reactor where the fermenting occurs. In some
embodiments,
3,5,7-trioxododecanoic acid or a salt of each thereof is exogenously supplied
to a reactor
where the fermenting occurs.
In another embodiment, the process further comprising separating the
hydrophobic phase from an aqueous phase, the separating comprising a first
continuous
centrifugation to separate the cells and the bulk of the spent broth from the
hydrophobic
phase, followed by a second continuous centrifugation to separate the
hydrophobic phase
from the remaining aqueous phase.
In another embodiment, the process further comprises isoprenylating the
divarin,
divarinic acid, or the divarinic acid acid ester included in the hydrophobic
phase, without
the need for a solvent swap, under conditions suitable to perform an
isoprenylation, to
prepare a cannabinoid or a mixture of cannabinoids.
In another embodiment, the divarinic acid or the salt thereof contained in the
hydrophobic phase is esterified with an alcohol under conditions suitable for
esterification
to provide alkyl divarinate.
In another embodiment, the alcohol utilized for esterification is selected
from
alcohols with 2 or more carbons such as 02-08 alcohols.
In another embodiment, the alkyl divarinate is reacted with an isoprenoid, or
is
isoprenylated, to produce a cannabinoid. In some embodiments, the reaction is
catalyzed
by a Bronsted acid. In some embodiments, the reaction is catalyzed by a Lewis
acid.
Examples of suitable catalysts include without limitation organic acids (e.g.
trifluoroacetic
acid, methanesulfonic acid, tosic acid, and the likes), mineral acids or
solutions of mineral
acids (e.g. hydrochloric acid, nitric acid, sulfuric acid, and the likes),
polymer-supported
acids (e.g. Amberlyst-1 5, polymer-supported tosic acid, Lewis acids (e.g.
BF3, Sc(0Tf)3,
and the likes), amino acids, or organocatalysts.
In another embodiment, the cannabinoid or the cannabinoid mixture comprises a
carboxyl moiety or a salt thereof, and is decarboxylated under conditions
suitable for
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decarboxylation, to prepare a decarboxylated cannabinoid. The decarboxylation
can be
modulated by heating the solution and/or by addition of a catalyst and/or by
the addition
of a base.
In another embodiment, the divarinic acid or the salt thereof contained in the
hydrophobic phase is decarboxylated to provide an initial composition
comprising divarin.
An acid may be added before or during decarboxylation to modulate the
decarboxylation
reaction. The reaction mixture may be heated to increase the decarboxylation
rate. A
base may be added to modulate the decarboxylation reaction.
In another embodiment, the initial composition comprising divarin is
isoprenylated
under conditions suitable for isoprenylating a phenolic compound. Examples of
compounds useful in isoprenylating include without limitation geraniol,
farnesol,
geranylgeraniol, p-mentha-2,8-dien-1-ol ((1 S,4R)-p-Mentha-2,8-dien-1 -01),
citral, and the
likes. In another embodiment, the isoprenylated compound is cannabigerovarin.
In
another embodiment, the isoprenylated compound is cannabigerovarinic acid.
In some embodiments, the initial composition comprising divarin is reacted
with
geraniol and an acid under conditions suitable to undergo a Friedel Crafts
alkylation to
provide cannabigerovarin (CBGV). The reaction may be performed in a suitable
solvent,
e.g., one that is inert to the reactants and the reagents. In some
embodiments, a Bronsted
acid is employed. In some embodiments, a Lewis acid is employed. The acid can
be
used in catalytic amounts. In some embodiments, the isoprenylated compound is
cannabigerovarin (CBGV). In some embodiments, the isoprenylated compound is
cannabigerovarinic acid (CBGVA). Suitable acids include a sulfonic acid, such
as p-
toluene sulfonic acid, BF3 etherate, and the likes. A skilled artisan will be
able to adapt
and modify, in view of this disclosure, known processes for preparing CBG and
CBGA for
preparing CBGV and CBGVA.
In some embodiments, the initial composition comprising divarin is reacted
with
citral under conditions suitable to undergo cyclization to provide
cannabichromevarin
(CBCV). In some embodiments, the isoprenylated compound is cannabichromevarin
(CBCV). In some embodiments, the isoprenylated compound is
cannabichromevarinic
acid (CBCVA). In some embodiments, citral reacts with divarin under basic
conditions to
provide cannabichromevarin. Suitable bases and other conditions will be
apparent to the
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skilled artisan upon reading this disclosure. Unreacted divarin can be
separated by
reacting with alkali.
In another embodiment, the cannabinoid composition is purified, optionally
hydrolyzed, and isolated to yield one or more cannabinoids. In another
embodiment, the
total cannabinoids contained in the isolated product is at least 25%, or 50%,
or 75%, or
90%, or 95%, or 98%, or 99% of a single cannabinoid.
In another embodiment, the cannabinoid is cannabigerovarinic acid (CBGVA). In
another embodiment, the cannabinoid is cannabichromevarinic acid (CBCVA). In
another
embodiment, the cannabinoid is cannabinovarinic acid (CBNVA).
In another
embodiment, the cannabinoid is tetrahydrocannabivarinic acid (THCVA). In
another
embodiment, the cannabinoid is cannabidivarinic acid (CBDVA). In another
embodiment,
the cannabinoid is cannabigerovarin (CBGV). In another embodiment, the
cannabinoid
is cannabichromevarin (CBCV). In another embodiment, the cannabinoid is
cannabivarin
(CBNV). In another embodiment, the cannabinoid is tetrahydrocannabivarin
(THCV). In
another embodiment, the cannabinoid is cannabidivarin (CBDV). In another
embodiment,
the cannabinoid is meroterpenoid compound that causes activation of the CB1,
CB2, or
TRP receptors.
In another embodiment, the illustrative and nonlimiting examples of an acyl-
CoA
includes Oleoyl-CoA, Palmitoleoyl-CoA, Stearoyl-CoA, Dehydrostearoyl-CoA,
Oxostearoyl-CoA, Enoyl-CoA, Oxacyl-CoA, Hexanoyl-CoA, Oxohexanoyl-CoA,
Butanoyl
(or ButyryI)-CoA, Crotonoyl-CoA, Acetoacetyl-CoA, Pentanoyl-CoA, or
Oxopentanoyl-
CoA.
In one embodiment, the acyl-CoA is a synthetic molecule that functions similar
to
an acyl-CoA and is accepted by the polyketide synthase enzyme. Non limiting
examples
of such synthetic molecules are provided, e.g., in Prasad, Gitanjeli et al. "A
mechanism-
based fluorescence transfer assay for examining ketosynthase selectivity.
"Organic &
biomolecular chemistry vol. 10,33 (2012): 6717-23. Incorporated herein by
reference.
In another embodiment, the polyketide synthase is olivetol synthase (OLS)
having
an amino acid sequence that is at least 95%, at least 96%, at least 97%, at
least 98%, or
at least 99% identical with SEQ ID 1. In another embodiment, the polyketide
synthase
shares at least 50% sequence identity with the amino acid sequence of SEQ ID 1
and
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whose alpha carbon backbone of its structure does not deviate by more than
1.5A with
(OLS) having the amino acid sequence of SEQ ID 1.
In another embodiment, the DABB protein is olivetolic acid cyclase (OAC)
having
an amino acid sequence that is at least 95%, at least 96%, at least 97%, at
least 98%, or
at least 99% identical with SEQ ID 2. In another embodiment, the DABB protein
has an
amino acid sequence that is at least at least 50% identical to olivetolic acid
cyclase (OAC)
of SEQ ID 2. In another embodiment, the OAC has an amino acid sequence at
least 95%,
at least 96%, at least 97%, at least 98%, or at least 99% identical with SEQ
ID 4, which
is described below.
Sequence ID 2: OAC/DABB
MAVKHLIVLKFKDE ITEAOKEEFFKTYVNLVN II PAMKDVYWGKDVTOKKEEGYTH IVEV
TFESV ETIOD YIIHP AHVGF GDVYR SFWEK LLIFD YTPRK
In another embodiment, the microorganism comprises an acyl-CoA synthetase
enzyme. Acyl-CoA refers, as is well known, to an ester of coenzyme A with a
carboxylic
acid. An acyl-CoA synthetase enzyme converts a carboxylic acid to an acyl-CoA.
In
another embodiment, the microorganism comprises an acyl-CoA synthetase enzyme,
which is CsAAE1 having an amino acid sequence of SEQ ID 3. In another
embodiment,
the microorganism comprises an acyl-CoA synthetase enzyme having an amino acid
sequence that is at least 50-75% identical with the amino acid sequence of SEQ
ID 3. In
another embodiment, at least a part of the acyl-CoA or a salt thereof is
exogenously
added to a reactor where the fermenting occurs. In another embodiment, the
acyl-CoA
like synthetic substrate or a salt thereof is exogenously added to a reactor
where the
fermenting occurs. In another embodiment, the carboxylic acid corresponding to
the acyl-
CoA or a salt thereof is exogenously added to a reactor where the fermenting
occurs.
Sequence ID 3: CsAAE1
MG KNYKSLDSVVASDFIALG ITSEVAETLHG RLAEIVCNYGAATPOT WIN IANH ILS PDL
PFSLHONALFYGCYKDFGPAPPAWIPDPEKVKSTNLGALLEKRGKEFLGVKYKDPISSF
SHFCEFSVRNPEVYWRIVLMDEMKISFSKDPECILRRDDINNPGGSEWLPGGYLNSA
KNCLNVNSNKKLNDTMIVWRDEGNDDLPLNKLTLDOLRKRVWLVGYALEEMGLEKGC
AIAIDMPMHVDAVVIYLAIVLAGYVVVSIADSFSAPEISTRLRLSKAKAIFTQDHIIRGKKRI
PLYSRVVEAKSPMAIVIPCSGSNIGAELRDGDISWDYFLERAKEFKNCEFTAREOPVDA
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YTNILFSSGTTGEPKAIPWTQATPLKAAADGWSHLDIRKGDVIVWPTNLGWMMGPWL
VYASLLNGASIALYNGSPLVSGFAKFVQDAKVTMLGVVESIVRSWKSTNCVSGYDWST
IRCFSSSGEASNVDEYLWLMGRANYKPVIEMCGGTEIGGAFSAGSFLOAGSLSSFSSQ
CMGCTLYILDKNGYPMPKNKPGIGELALGPVMFGASKTLLNGNHHDVYFKGMPTLNG
EVLRRHGDIFELTSNGYYHAHGRADDTMNIGGIKISSIEIERVCNEVDDRVFETTAIGVP
PLGGGPEOLVIFFVLKDSNDTTIDLNQLRLSFNLGLQKKLNPLFKVTRVVPLSSLPRTAT
NKIMRRVLRQQFSHFE
In one embodiment, the hydrophobic phase utilized herein comprises an alkane.
In one embodiment, the hydrophobic phase comprises an alcohol preferably with
carbon
number greater than 4 such as a C5-C8 alcohol. In one embodiment, the
hydrophobic
phase comprises an ester. In one embodiment, the hydrophobic phase comprises a
triglyceride. In one embodiment, the hydrophobic phase comprises a diester
such as
dialkyl malonate. In one embodiment, the hydrophobic phase comprises a
commercially
available oil. Examples of such oils include without limitation sunflower oil,
olive oil,
vegetable oil or the like). In one embodiment, the hydrophobic phase
comprises a
combination of the various hydrophobic phases disclosed hereinabove.
Methods for prenylating olivetol, olivetolic acid, olivetolc acid esters,
divarin,
divarinic acid, divarinic acid esters, and such other resorcinol derivatives
utilized herein,
e.g., and without limitation, with geraniol, citral, cyclic isoprenoids, and
the likes are known
in the art (see e.g., W02019033168, US2017/283837, US2015/336874,
US2018/244642,
US2009/36523, W02010/59943, US4315862, each of which is incorporated herein in
its
entirety by reference), and can be modified based on the disclosure provided
herein by a
skilled artisan.
The biosynthesis of certain illustrative and non-limiting cannabinoids, as
utilized
herein, is described below.
The scheme below illustrates aromatic polyketides (1A), ketones (Figure 1B)
and
lactones (Figure 10) and other diverse class of chemical compounds that have a
wide
range of applications in the industrial and.
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OH 0 0
HO 0 R2 A....I
I A,
HO R3 HO
Ri
A B C
OH OH OH
CO2H 0 0 CO2H
HO HO HO
D E F
0 0
0 0
HO HO
G H
General examples of the production of acid or non-acidic polyketides (IA), the
production of
ketones (113), and the production of lactones (IC). Specific examples are the
production of the
polyketide olivetol (ID), olivetolic acid (1E), the lactone PDAL (1G) and a
ketone HTAL (IH). For
example, and not for limitation, all products are produced using a polyketide
synthase olivetol
synthase (OLS); other suitable TKS enzymes are also useful. To make olivetolic
acid a DABB
protein, here olivetolic acid cyclase (OAC) is co-expressed with the
polyketide synthase to
produce the acidic polyketide. ID, 1E, IG, and IH are formed, for example,
from malonyl-CoA and
hexanoyl-CoA while F is form from malonyl-CoA and butyl-CoA.
In one embodiment, a compound of formula ID is provided. In one embodiment, a
compound of formula IE or a salt thereof is provided. In one embodiment, a
compound of
formula IF or a salt thereof is provided. In one embodiment, a compound of
formula IG is
provided. In one embodiment, a compound of formula IH is provided.
In some embodiments, provided herein are biosynthetic cannabinoids, as
distinct
from cannabinoids made by chemical synthesis only, where such biosynthetic
cannabinoids comprise trace or tell-tale amounts (less than 3%, preferably
less than 2%,
more preferably less than 1%) of fermentation derived byproducts. In some
embodiments,
the byproduct is a lactone of formula IC, such as FOAL.
These compounds are produced by many natural sources and represent a
valuable class of natural products. In nature many different plants and
microorganisms
produce these types of compounds. These compounds can be functional molecules
themselves or are used to created more complex compounds through additional
chemical
steps such as prenylation or esterification. In nature aromatic polyketides,
ketones, and
lactones are formed from the combination of 2 or more malonyl-CoA molecules
and also
typically involve an additional substrate such as an acyl-CoA molecule. Herein
we
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disclose a method to produce a diverse set of polyketides, ketones, and
lactones using
engineered microorganisms. We also describe a method wherein these compounds
can
be extracted and further processed to create additional molecules of value
such as
cannabinoids.
The invention described below focuses on the production of aromatic
polyketides,
ketones and lactones from yeast, bacteria, and/or algae. The invention can be
used to
create diverse chemical libraries for therapeutic screening or as an
industrial scale
production platform for high value natural compounds. In one aspect of the
invention
diverse chemical compounds can be form by supplementing the media surrounding
engineered cells with different acyl-CoA compounds or different acyl-acid
compounds
which are then transformed into acyl-CoA compounds in vivo. In another aspect
of the
invention the aromatic polyketides, ketones and/or lactones can be extracted
from the
media using an immiscible hydrophobic layer that is added to the fermentation
vesicle. In
another aspect of the invention these aromatic polyketides, ketones and/or
lactones, once
produced and collected, can be used to create more complex chemical components
through the chemical reactions such as but not limited to prenylation or
esterification.
In another embodiment, the OAC utilized herein has Sequence ID 4:
MAVKHLIVLKFKDE ITEAQKEEFFKTYVNLVN II PAMKDVYWGKDVTQKNKEEGYTH IVE
VTFESVETIQDYIIHPAHVGFGDVYRSFWEKLLIFDYTPRK
In Vivo Production of aromatic polyketides, ketones and/or lactones
The aromatic polyketides, ketones and lactones of interest are formed from
fatty
chain acyl-CoA and condensation of malonyl-CoA. In order to produce these
compounds,
the enzyme that condense these compounds together (a polyketide synthase) must
take
at least 2 malonyl-CoA. There are many different polyketide synthase enzymes
that
perform this function. The choice of enzyme used must be able to be expressed
in the
host cell and function appropriately. One enzyme in particular, the polyketide
synthase
from the cannabis plant (OLS SEQ ID 1), can be used to create these different
compounds. In this invention this enzyme has been functionally expressed into
yeast in
order to produce various polyketide products. This enzyme can also be
expressed in
bacteria and used in vitro to produce various polyketide, ketone, or lactone
products. The
same enzyme can be used to create either a lactone, ketone, or polyketide with
the
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different between the final products having to do with the number of malonyl-
CoAs
involved and if/or when the product is hydrolyzed off. Lactones require 2
malonyl-CoAs
while the ketones and polyketides required 3 malonyl-CoAs. Controlling what
compound
is formed can be done through engineering of the polyketide synthase enzyme,
limiting
availability of malonyl-CoA, or increasing the speed of hydrolysis. In some
embodiments
of this invention increasing or decreasing the availability of malonyl-CoA can
lead to
different product formations.
In addition to requiring malonyl-CoA the enzyme can also use acyl-CoA as
substrates which leads to a diversity in the chemical products. There are
several aryl-
CoAs that act as substrates for the enzyme including but not limited to:
Oleoyl-CoA,
Palmitoleoly-CoA, Stearoyl-CoA, Dehydrostearoly-CoA, Oxostearoyl-CoA, Enoyl-
CoA,
Oxacyl-CoA, Hexanoyl-CoA, Oxohexanoyl-CoA, Butanoyl-CoA, Crotonoyl-CoA,
Acetoacetyl-CoA, Pentanoyl-CoA, Oxopentanoyl-CoA. Synthetic molecules that
have the
same function as a CoA could also be used as substrates leading to increased
chemical
diversity. There are several ways in which various CoAs can be made. The
production of
fatty chain acyl-CoA or other specialized CoA containing compounds can be
initiated in a
variety of ways. In one embodiment enzymes can be introduced and overexpress
to
produce these compounds directly from sugar. There are several examples of the
enzymes that are responsible for the production of fatty chain acyl-CoA are.
In an
alternative approach, fatty chain acyl-CoA can be produced by supplementing
the growth
media with a fatty acid and then incorporating a CoA charging enzyme. There
are several
examples of enzyme that can produce these types of CoAs including CsAAE1 (SEQ
ID
2). Once the -CoA compound is formed it can be acted on by the polyketide
synthase
which results in different products being form.
In another aspect of the invention acidic polyketides can be made. These
acidic
polyketides can have unique therapeutic properties, such as novel antibiotics.
In many
cases the terminations and release of the polyketide product from the
polyketide synthase
results in its decarboxylation. In one aspect of this invention mixtures of
acidic and non-
acidic polyketides can be made by including an additional dimeric a-pl3 barrel
(DABB)
protein. When this DABB protein is co-expressed with the polyketide synthase
the
resulting polyketide will have a carboxylic acid group. In one embodiment of
this invention
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the dimeric a+13 barrel (DABB) protein is the olivetolic acid cyclase enzyme
which is found
in the cannabis plant (OAC SEQ ID 3).
Extraction of aromatic polyketides, ketones and lactones
In one aspect of the invention the extraction of the aromatic polyketides,
ketones
and/or lactones can be done In situ through the use of an immiscible organic
solvent or
oil layer. In order to achieve high product titers, the microorganism that is
expressing the
enzymes to produce the aromatic polyketides, ketones and/or lactones is made
to secrete
the products to the media; secretion can lead to high product titers. Often
these products
are not very water soluble or are toxic to the microorganisms themselves. Real-
time
removal of the product eliminates this toxicity as well as adds in
streamlining further
downstream processing. The choice of oil must have the following properties:
the
aromatic polyketides, ketones and lactones (the products) are soluble in this
layer, the
layer is immiscible with water, the layer is not toxic to the microorganism
itself. Examples
of useful extractions layers are dodecane and isopropyl myristate. Ideally the
oil layer
chosen has a high solubility for the products of interest and a low solubility
for various
media components such as sugars and vitamins to minimize additional downstream
purification.
During fermentation or cell growth the oil layer is added to the growth
vesicle. The
compounds of interest that are produced are excreted from the cells and then
collected
in the immiscible oil layer creating a chemical sink for the product. This
procedure extracts
the compounds as well as minimizes their toxicity to the cells in solution.
After
fermentation has concluded the oil is separated from the growth media. There
are several
ways to separate the oil from the media and one such method would be
centrifugation. In
this example the oil is separated from the media by centrifugation, the oil is
collected
which contains the compounds of interest.
Modification of aromatic polyketides, ketones and lactones
In one aspect of this invention after the aromatic polyketides, ketones or
lactones
are produced by the microorganism and are collected in the oil layer
subsequent chemical
reactions on the aromatic polyketides, ketones or lactones can occur.
Additional
modifications can be the dimerization of acidic polyketides, the
esterification of acidic
polyketides or the prenylation of polyketides, ketones or lactones or a
combination of
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these chemical transformations through chemical or enzymatic means.
Prenylation of the
aromatic ring can lead to additional compounds that have the polyketide,
lactone or
ketone as their backbone. For the chemical synthesis of either prenylated
products or
other products the preferred reaction is with a molecule that contains a
hydroxy group.
Examples of these types of prenyl groups that could be attached to the
aromatic ring
would be farnesol, geraniol, prenol, citronellol, or 2-Methyl-3-buten-2-ol.
For example, the
addition of geraniol to olivetol creates cannabigerol (CBG), which is a
natural
cannabinoid. Other compounds can be added to the products derived from
fermentation
such as (1S,4R)-p-Mentha-2,8-dien-1-ol which results in the formation of
cannabidiol or
tetrahydrocannabidiol like molecules.
In some aspects of this invention it is preferable to use an oil layer that is
compatible with subsequent chemical reactions. The choice of the oil used
should follow
the criterion described above and it is also preferable to choose an oil that
allows for
chemical reactions to take place. In some aspect of the invention the choice
of oil is not
compatible with additional chemical reactions. In this case the compounds must
first be
extracted from the oil layer and then reconstituted into a solvent that will
allow for further
chemical manipulations.
An illustrative and non-limiting process of isolating olivetol or another
aromatic
polyketide is schematically illustrated in Figure 1.
In one embodiment, a mixture of compounds of an aromatic polyketide, the
polyketide carboxylic acid, or a salt thereof provided by fermentation is
extracted from a
fermentation media by alkaline extraction. In some embodiments, the alkaline
extraction
is an aqueous alkaline extraction. In some embodiments, the alkaline
extraction is
performed at a pH of about 12 - about 14. In some embodiments, the alkaline
extraction
is performed at a pH of about 13.
In some embodiments, the extracted mixture of compounds of an aromatic
polyketide, the polyketide carboxylic acid, or a salt thereof are
decarboxylated to provide
the aromatic polyketide. In some embodiments, the decarboxylation is performed
by
heating. In some embodiments, the heating is performed at about 100 C ¨ about
140 C,
or preferably at about 110 C ¨ about 130 C. In some embodiments, the heating
is
performed at about 120 C. Post decarboxylation, the aromatic polyketide
provided,
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comprises by weight about 2% or less, or preferably about 1% or less of the
polyketide
carboxylic acid, or a salt thereof. In some embodiments, the extracted mixture
of an
aromatic polyketide, the polyketide carboxylic acid, or a salt thereof are
acidified before
decarboxylation. In some embodiments, the decarboxylation is performed at a pH
of
about 5 - about 8. In some embodiments, the decarboxylation is performed at a
pH of
about 6.5.
In one embodiment, aromatic polyketide provided by decarboxylation is
extracted
into an organic solvent (e.g., a water immiscible organic solvent) to provide
a solution of
the compound of formula IA in the organic solvent. In some embodiments, the
organic
solvent is a solvent capable of dissolving a compound of the aromatic
polyketide; the
aromatic polyketide comprising an aromatic ring and polar hydroxy groups. In
one
embodiment, the organic solvent comprises an aromatic hydrocarbon solvent. In
one
embodiment, the organic solvent comprises toluene. In one embodiment, the
organic
solvent is toluene. In some embodiments, the organic solvent comprises
aliphatic or
alicyclic hydrocarbon solvents.
In some embodiments, the aromatic polyketide, present as a solution in the
organic
solvent, is reacted with a terpene alcohol, a terpenal (i.e., a terpene
aldehyde), and the
likes. In some embodiments, the solution of the aromatic polyketide in the
organic solvent
is employed for reacting the aromatic polyketide with a terpene alcohol. In
some
embodiments, the solution of the aromatic polyketide in the organic solvent is
employed
for reacting the compound the aromatic polyketide with a terpenal. In one
embodiment,
the terpine alcohol is geraniol. In one embodiment, the terpene alcohol is
farnesol. In one
embodiment, the terpene alcohol is menthadienol (trans 2,8-menthadienol or
(15,4R)-p-
Mentha-2,8-dien-1-01). In one embodiment, the terpene alcohol is trans 2,8-
menthadienol.
In one embodiment, the terpene alcohol is (1S,4R)-p-Mentha-2,8-dien-1-o1). In
one
embodiment, the terpenal is citral. In some embodiments, the reaction with a
terpenal
further comprises a primary amine. In one embodiment, the primary amine is
tertiary
butyl amine.
In some embodiments, the reaction of the aromatic polyketide with a terpene
alcohol, a terpenal, or the likes provides a cannabinoid. In one embodiment,
the
cannabinoid is cannabigerol (CBG). In another embodiment, the cannabinoid is
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cannabichromene (CBC). In another embodiment, the cannabinoid is cannabidiol
(CBD).
In another embodiment, the cannabinoid is tetrahydrocannabinol (THC). In
another
embodiment, the cannabinoid is cannabinol (CBN). In another embodiment, the
cannabinoid is the varin analog (CBGV, CBCV, CBDV, THCV, CBNV) of CBG, CBC,
CBD, THC, CBN. A varin analog is a compound where the n-pentyl chain of a
cannabinoid, e.g., and without limitation, CBG, CBC, CBD, or THC is replaced
by an n-
propyl chain. The cannabinoids obtained are purified by a variety of
purification methods.
In one embodiment, the purification method comprises chromatography. In one
embodiment the purification method comprises distillation. In one embodiment,
the
chromatography comprises a reverse phase chromatography.
In one embodiment, the aromatic polyketide is olivetol. In another embodiment,
the
aromatic polyketide is divarin.
A non-limiting example of reacting (prenylating) olivetol with the terpene
alcohol,
geraniol, is schematically illustrated in Figure 2. A non-limiting example of
reacting
(prenylating) olivetol with the terpenal, citral, is schematically illustrated
in Figure 3..
EXAMPLES
These illustrative and non-limiting examples can be adapted according to the
present disclosure to provide the methods and compositions provided herein.
EXAMPLE I: Preparation of Cannabichromene
To a three-necked round bottomed flask (100 ml capacity), fitted with a
dropping
funnel and a condenser is added 5 g olivetol (27.8 mmole) and 2.03 g (2.96 ml,
27.8
mmole) t-butyl amine in 55 ml toluene and the mixture is heated to 50 -60 C,
4.23 g (4.76
ml, 27.8 mmole) of citral is then added dropwise. The mixture is refluxed for
9 hours, after
which time it is cooled to room temperature and the solvent evaporated to give
a crude
reaction mixture.
EXAMPLE II: Purification of Cannabichromene
g of the crude reaction mixture from Example I is dissolved in 100 ml toluene
and
the solution extracted twice with 50 ml of 1% aqueous sodium hydroxide
solution followed
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by 50 ml of water. The toluene solution is then dried over anhydrous sodium
sulfate and
the solvent evaporated. The residue is then dissolved in 50 ml ethanol, and
250 mg of
sodium borohydride are added portion-wise while stirring. Stirring at room
temperature is
continued for 30 minutes after which time the solvent is evaporated and the
residue
partitioned between water (50 ml.) and toluene (100 ml). The crude reaction
mixture is
chromatographed on a column of processed silica gel (200 g). Processed silica
gel is
prepared by making a paste of silica gel -PF254 with water (equal amount)
which is then
dried in an oven at 110 C and the resulting cake passed through 60 mesh sieve.
The
solvent system used is a mixture of toluene and chloroform (1:1). Fractions
are collected
and the solvent evaporated to provide pure CBC.
Olivetol utilized in the examples provided herein can be replaced by other
alkyl
resorcinols, such as, 5-propy1-1,3-dihydroxybenzene or divarin to prepare
"varin" and
such other analogs of CBC such as CBCV and CBCVA. Citral utilized in the
examples
provided herein can be replaced by other terpene aldehydes such as farnesal to
prepare
isoprene homologs of CBC.
EXAMPLE Ill: Preparation of Cannabigerol (CBG)
Oliveto! (2 g) and geraniol (3 g) are dissolved in 400 ml of chloroform
containing p-
toluenesulfonic acid (80 mg) and stirred at room temperature for 12 h in the
dark.
Chloroform may be replaced by toluene, cyclohexane, and such other solvents.
The
reaction mixture is washed with 400 ml of saturated sodium bicarbonate and
then with
400 ml of water. After the chloroform layer is concentrated at 40 C under
reduced
pressure, the residue is chromatographed on a 2.0 x 25-cm column of silica
gel. The
column is eluted with 1000 ml of toluene to give CBG (1.4 g).
Olivetol utilized in the examples provided herein can be replaced by other
alkyl
resorcinols, such as, 3-propy1-1,5-dihydroxybenzene to prepare "varin" and
such other
analogs of CBG. Geraniol utilized in the examples provided herein can be
replaced by
other terpenols such as farnesol to prepare isoprene homologs of CBG.
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EXAMPLE IV: Production of Cannabigerol (CBG)-10L scale
Oliveto! (335 g) and geraniol (574 g) are dissolved in 5,500 g of toluene
containing
p-toluenesulfonic acid monohydrate (42.5 g) and stirred at 30 C for 1.5 hour
in a 10 L
jacketed reactor. The reaction mixture is quenched with 700 ml of saturated
sodium
bicarbonate. After 30 minutes, agitation is stopped to allow phase separation.
The
aqueous layer is separated and discarded as waste. The organic layer is then
washed
with 2.7 L of DI-water for 30 minutes. After draining the aqueous layer, the
organic layer
is concentrated at 50 C under reduced pressure to 150 g/L of CBG. The residue
is
chromatographed on a spherical silica gel column with a particle size
distribution of 40-
75 m. The column is eluted with toluene and ethyl acetate gradient to purify
the CBG
from other impurities. A typical gradient is as follow:
Step Ethyl Acetate Start Ethyl Acetate End Length
Equilibration 0% 0% 0.20 CV
1 0% 0% 1.00 CV
2 0% 20% 2.00 CV
3 20% 20% 2.00 CV
4 20% 80% 1.00 CV
Toluene can be replaced by hexane, heptane, and such other solvents. Ethyl
acetate can be replaced by 2-propanol or acetone.
Olivetol utilized in the examples provided herein can be replaced by other
alkyl
resorcinols, such as, 3-propy1-1,5-dihydroxybenzene to prepare "varin" and
such other
analogs of CBG. Geraniol utilized in the examples provided herein can be
replaced by
other terpenols such as farnesol to prepare isoprene homologs of CBG.
EXAMPLE V: Production of Cannabigerol (CBG)
A. Oliveto! (4.538 kg) is dissolved in 83 L of toluene containing p-
toluenesulfonic
acid monohydrate (0.359 kg) and stirred at 30 C in a jacketed reactor.
Geraniol (5.825
kg) is charged to the reactor and stirred for 1 h. The reaction mixture is
quenched with
saturated sodium bicarbonate (6.625 kg) and cooled to 15 C. After 30 minutes,
agitation
is stopped to allow phase separation. The aqueous layer is separated and
discarded as
waste. DI water (20 L) is charged to the reactor and mixed for 30 minutes. The
aqueous
layer is separated and discarded as waste. The organic solution is
concentrated under
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vacuum to yield crude CBG concentrate (6.06 kg). The crude CBG concentrate is
purified
by liquid chromatography on alumina media with toluene as eluent. The purified
CBG is
concentrated under vacuum to yield purified CBG concentrate (2.25 kg).
B. Purified CBG concentrate (9 kg) is dissolved in n-heptane (13.8 kg) and
cooled
slowly to -10 C. The product slurry is filtered and washed with cold n-
heptane (6.2 L).
The product cake is dried under N2 to give 3.36 kg CBG.
C. CBG crystals (3.36 kg) are dissolved in n-heptane (44.7 L) under N2 at 40
C.
The solution is cooled slowly to 28 C and held for 30 minutes. The solution is
cooled
slowly to 5 C and then held for 1 h. The product slurry is filtered and washed
with cold
heptane (6 L). The product cake is dried under N2 to give pure CBG crystals
(2.18 kg).
EXAMPLE VI: Preparation and Purification of Cannabichromene
To a three-necked round bottomed flask (5L capacity) equipped with a condenser
under N2 atmosphere is added 106.3 g olivetol (0.59 mol) in 1.86 L o-xylene
and the
mixture is heated to 45 C. At 45 C solution temperature, 134.71 g citral (0.88
mol) and
21.56 g t-butylamine (0.29 mol) are charged to the vessel. The mixture is
heated to 1300
and hold for 2.5 hours, after which time it is cooled down to room temperature
and
quenched with 0.35L of 1M phosphoric acid. After 15 minutes, agitation is
stopped to
allow phase separation. The aqueous layer is separated and discarded as waste.
The
organic layer is then washed with 0.35L of DI water, which is drained after 15
minutes.
The organic layer is concentrate at 70 C under full vacuum to 500g/L of CBC.
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